Mesenchymal stem cells attenuate ischemic acute kidney injury by inducing regulatory T cells through splenocyte interactions

Abstract

The mechanism of mesenchymal stem cell therapy in acute kidney injury remains uncertain. Previous studies indicated that mesenchymal stem cells could attenuate inflammation-related organ injury by induction of regulatory T cells. Whether regulatory T-cell induction is a potential mechanism of mesenchymal stem cell therapy in ischemic acute kidney injury and how these induced regulatory T cells orchestrate local inflammation are unknown. Here we found that mesenchymal stem cells decrease serum creatinine and urea nitrogen levels, improve tubular injury, and downregulate IFN-γ production of T cells in the ischemic kidney. In addition to the lung, mesenchymal stem cells persisted mostly in the spleen. Mesenchymal stem cells increased the percentage of regulatory T cells in the spleen and the ischemic kidney. Antibody-dependent depletion of regulatory T cells blunted the therapeutic effect of mesenchymal stem cells, while coculture of splenocytes with mesenchymal stem cells caused an increase in the percentage of regulatory T cells. Splenectomy abrogated attenuation of ischemic injury, and downregulated IFN-γ production and the induction of regulatory T cells by mesenchymal stem cells. Thus, mesenchymal stem cells ameliorate ischemic acute kidney injury by inducing regulatory T cells through interactions with splenocytes. Accumulated regulatory T cells in ischemic kidney might be involved in the downregulation of IFN-γ production.

Figure 1

Mesenchymal stem cells (MSCs) ameliorate renal ischemia/reperfusion injury (IRI). In comparison with animals treated with saline, infused MSCs significantly reduced serum creatinine (SCr (a)) and blood urea nitrogen (BUN (b)) 24 h and 72 h after IRI. IRI could induce severe tubular injury (e). Moreover, MSC therapy markedly improved tubular injury (f) and reduced acute tubular necrosis (ATN) scores (c) 72 h after IRI. Sham-operated control (sham, d) had no effect on renal histopathological parameters (c and d). For SCr (a) and BUN (b), values are mean±s.e., n=8–10 in each group. *P<0.05 vs. sham, ^#P<0.05 vs. IRI treated with saline. For ATN scores (c), values are mean±s.e. Approximately 80 high-power fields (HPFs, × 400) per individual mouse (20 HPFs per slide, four slides per animal) were evaluated. n=6 in each group. ^#P<0.05 vs. IRI treated with saline.

Figure 2

Mesenchymal stem cells (MSCs) suppress ischemia/reperfusion injury (IRI)–induced upregulation of proinflammatory cytokine IFN-γ. IRI per se could only slightly increase the number of CD8⁺(B)T cells infiltrating in ischemic kidney after 72 h of reperfusion. MSCs had no roles in the number of CD4⁺ (a) and CD8⁺ (b) T cells infiltrating in ischemic kidney both after 24 and 72 h of reperfusion. In comparison with animals treated with saline, MSCs could reduce IFN-γ production of CD4⁺ (c) and CD8⁺ (d) T cells infiltating in ischemic kidney after both 24 and 72 h of reperfusion. The number and IFN-γ production of CD4⁺ and CD8⁺ T cells were measured by flow cytometry. For the percentage of IFN-γ-positive cells in CD4⁺ and CD8⁺ T cells, values are mean±s.e., n=5 in each group. *P<0.05 vs. sham, ^#P<0.05 vs. IRI treated with saline.

Figure 3

Infused mesenchymal stem cells (MSCs) persist in spleen after ischemia/reperfusion injury (IRI). Intravenously delivered RFP-labeled MSCs were not detected in ischemic kidney at any time point (a), but persisted in spleen during the whole process, at least for 120 h after reperfusion (b). RFP-labeled MSCs (red) were detected by laser confocal microscopy. Nuclei were stained with DAPI (blue). Original magnification, × 600, n=3. (c) Intravenously delivered DiR-labeled MSCs (yellow and red) persisted in the spleen at least for 120 h after reperfusion. Signals were detected by IVIS in vivo imaging, n=3.

Figure 4

Mesenchymal stem cells (MSCs) increase the percentage of CD25⁺Foxp3⁺ cells in CD4⁺ T cells in ischemic kidney and spleen. Ischemia/reperfusion injury (IRI) per se increased the percentage of CD25⁺Foxp3⁺ cells in CD4⁺ T cells (a and b) in ischemic kidney after 72 h of reperfusion. In comparison with animals treated with saline, MSCs increased the percentage of CD25⁺Foxp3⁺ cells in CD4⁺ T cells (a and b) after 72 h of reperfusion. IRI per se had no role in CD25⁺Foxp3⁺ cells in CD4⁺ T cells in the spleen (c and d) after 72 h of reperfusion. In comparison with animals treated with saline, MSCs increased the percentage of CD25⁺Foxp3⁺ cells in CD4⁺ T cells (c and d). The percentage of CD25⁺Foxp3⁺ cells in CD4⁺ T cells was measured by flow cytometry. For the percentage of CD25⁺Foxp3⁺ cells in CD4⁺ T cells, values are mean±s.e. n=5 in each group. *P<0.05 vs. sham, ^#P<0.05 vs. IRI treated with saline.

Figure 5

Depletion of Tregs blunts renoprotection of mesenchymal stem cells (MSCs). In mice with IgG injection, MSCs could significantly improve serum creatinine (SCr (a)), blood urea nitrogen (BUN (b)), and tubular injury (c–e) 72 h after IRI compared with saline-treated mice. In mice with PC61 injection, MSCs only slightly improved SCr (a), BUN (b), and tubular injury (c, f and g) 72 h after IRI in mice with saline-treated mice. However, in MSC-treated mice, animals with PC61 injection still showed higher levels of SCr (a) and BUN (b) and aggravation of tubular injury (c, e, and g) 72 h after IRI compared with mice with IgG injection. For SCr (a) and BUN (b), values are mean±s.e. n=5 in each group. For ATN scores (c), approximately 80 high-power fields (HPFs, × 400 ) per individual mouse (20 HPFs per slide, four slides per animal) were evaluated. Values are mean±s.e., n=5 in each group. ^$P<0.05 vs. IRI+IgG, ^*P<0.05 vs. saline-treated mice with PC61 injection; ^#P<0.05 vs. MSC-treated mice with IgG injection.

Figure 6

Coculture with mesenchymal stem cells (MSCs) increases the percentage of Tregs in splenocytes. The percentage of CD25⁺Foxp3⁺ cells in CD4⁺ T cells was detected by flow cytometry. The percentage of Tregs significantly increased after coculture with MSCs for 72 h (a and b). The IFN-γ production by CD4⁺ and CD8⁺ T cells in splenocytes stimulating with PMA/Ionocymin was detected by intracelluar staining via flow cytometry. Splenocytes removed from the coculture system displayed a significantly lower capacity of IFN-γ secretion of both CD4⁺ (c and d) and CD8⁺ T cells (d and e). Proliferation of splenocytes stimulated by ConA (10 μg/ml) was detected by WST-8 kit. Splenocytes removed from the coculture system displayed a significantly lower proliferation rate (f) after stimulation. For the percentage of CD25⁺Foxp3⁺ cells in CD4⁺ T cells (a and b), values are mean±s.e. n=5 in each group. *P<0.05 vs. splenocytes culture alone. For the percentage of IFN-γ-positive cells in CD4⁺ and CD8⁺ T cells, values are mean±s.e. n=5 in each group. *P<0.05 vs. splenocyte culture alone. For the proliferation of splenocytes, values are mean±s.e., n=5 in each group. *P<0.05 vs. splenocyte culture alone; ^#P<0.05 vs. splenocytes stimulated by ConA.

Figure 7

Splenectomy abolishes mesenchymal stem cell (MSC)–induced renoprotection. In ischemia/reperfusion injury (IRI) mice with splenectomy, infused MSCs had no roles in serum creatinine (SCr (a)), blood urea nitrogen (BUN (b)), and tubular injury (e–g) 72 h after IRI. MSC therapy showed no reduction of IRI-induced upregulation of IFN-γ of CD4⁺ and CD8⁺ T cells (c) 72 h after IRI. Moreover, MSC treatment showed no effect on the percentage of Tregs in ischemic kidney (d) 72 h after IRI. The IFN-γ production of CD4⁺ and CD8⁺ T cells and the percentage of Tregs were measured by flow cytometry. For SCr (a) and BUN (b), values are mean±s.e., n=6 in each group. For the percentage of IFN-γ-positive cells in CD4⁺ and CD8⁺ T cells (c), values are mean±s.e., n=5 in each group. For the percentage of CD25⁺Foxp3⁺ cells in CD4⁺ T cells (d), values are mean±s.e. n=5 in each group. For ATN scores (g), ∼80 high-power fields (HPFs, × 400 ) per individual mouse (20 HPFs per slide, four slides per animal) were evaluated. Values are mean±s.e. n=5 in each group.